PROJECT SUMMARY In collaboration with Dr. Wilen (Yale University) and Dr. Goujon (CNRS, France), we will establish cell line models and conduct genome-wide CRISPR screens to identify host genes that are necessary for SARS-CoV-2 infection. We will first use Vero cells from the African Green Monkey, as they are a well-established model for many pathogens, and we had previously generated a genome-wide library for this species. Preliminary data suggest that we have also generated human cell lines that can serve as effective model systems, and these screens will be conducted as soon as the models are sufficiently validated. Across these screens, we expect to find host factors that are necessary for infection, such as the surface binding target for the virus, ACE2, and anticipate that the screens will identify additional genes that, when knocked out, prevent viral cytopathic effects. Additionally, we can modulate the selective pressure to identify factors that, when lost, sensitize the cells to SARS-CoV-2, which will provide a complementary view into host cell biology. Likewise, we will also conduct CRISPR activation screens to identify host genes that, when overexpressed, provide protection against infection, which may identify restriction factors that the virus must overcome. Primary screens will be validated with secondary pools, which will also allow for testing of genes across more conditions and cell types, establishing the generalizability of the results. Finally, combinatorial screens will be conducted to generate unbiased genetic interaction maps of hit genes, which can identify redundancies that are partially masked in a primary screen, as well as unexpected synergies across pathways. Focused, mechanistic follow-up of genes identified by these screens is outside the scope of this proposal, but is of immediate interest to the Wilen and Goujon groups.